Heterobifunctional compounds having the general formula I (shown below) are well known as coupling agents that are useful for coupling chemical compounds. ##STR2## Some of the most commonly used coupling agents include: m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfosuccinimidyl 4-(p-maleimidophenyl) butyrate (S-SMPB), m-maleimidobenzoylsulfosuccinimide ester (S-MBS) and N-.gamma.-maleimidobutyryloxysuccinimide ester (GMBS).
Homobifunctional compounds have also been found useful as coupling agents. Examples of such compounds can be found in co-pending U.S. patent application Ser. No. 07/600,795 now abandoned which enjoys common ownership with the instant application.
Compounds having the general formula I have two reactive groups which serve as the basis for the utility of these compounds as coupling agents. The first reactive group is the N-hydroxysuccinimide group (NHS) or a water soluble analog thereof. At an alkaline pH, amino groups displace the NHS by nucleophilic attack of the ester. A reaction between a compound of formula I and an amine functional compound results in the acylation of the amino group of the amine functional polymer.
The second reactive group is the maleimide group which is reactive at the carbon-carbon double bond. In particular, at a neutral pH, the maleimide moiety is reactive at the carbon-carbon double bond with thiol groups, and such a reaction results in the formation of a thioether bond.
Presently, coupling two compounds using a compound of the formula I takes place in a stepwise manner and, generally, is performed in a two step process. The first step of the process takes place at a neutral or weakly alkaline pH and links an amine functional compound to a coupling agent as outlined above. The second step of the process takes place at a neutral pH and couples a thiol functional compound to the coupling agent in the manner previously stated. Hence, the amine functional compound and the thiol functional compound are coupled.
The method outlined above has been used to couple a variety of compounds. For example: Kitagawa et al., J. Biochem., 79, 233-236 (1976). used MBS to couple insulin with .beta.-D-galactosidase; Meyers, D. T. et al., J. Immunol. Methods, 121, 129-142 (1989), used MBS, S-MBS, GMBS and S-SMPB to couple an antibody to ricin (a toxin) to produce a immunotoxin (IT), and Dell'Arciprete L. et al., J. Immunol., 140, 2466-2471 (1988), used MBS to conjugate an antibody to diphtheria toxin to form an IT.
Although the aforementioned coupling method has been used successfully, it is limited in the types of compounds it can couple. More specifically, in order to successfully couple two compounds, the coupling method requires an amine functional compound and a thiol functional compound. If, for example, it were desired to couple two proteins (neither of which had a thiol group) it would be necessary to introduce a thiol group to one of the proteins in order to use the coupling method currently available. However, adding a thiol group to a compound may induce unwanted conformational changes to the compound and the removal of excess thiolating reagent is mandatory before the compound is coupled. Additionally, thiol groups are less stable at ambient conditions than, for instance, amine groups. Due to the necessity for a thiol group in the second step of the previously mentioned process, that process is limited in its application.
There is thus a need for a coupling process that does not require compound modification prior to coupling.